MARINE ECOLOGY PROGRESS SERIES Vol. 270: 83–102, 2004 Published April 14 Mar Ecol Prog Ser Photosynthetic pigments in 37 species (65 strains) of Haptophyta: implications for oceanography and chemotaxonomy Manuel Zapata1, S. W. Jeffrey2,*, Simon W. Wright3, Francisco Rodríguez1, José L. Garrido4, Lesley Clementson2 1Centro de Investigacións Mariñas (CIMA), Consellería de Pesca, Xunta de Galicia, Apartado 13, 36620 Vilanova de Arousa, Spain 2CSIRO Marine Research, GPO Box 1538, Hobart, Tasmania 7001, Australia 3Australian Antarctic Division, Kingston, Tasmania 7050, Australia 4Instituto de Investigacións Mariñas, Consejo Superior de Investigacións Cientificas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain ABSTRACT: The pigment compositions of 37 species (65 strains) of cultured haptophytes were analysed using improved HPLC methods. We distinguished 8 pigment types based on the distribution of 9 chlorophyll c (chl c) pigments and 5 fucoxanthin derivatives. All types contained chl c2 and Mg- 2,4-divinyl phaeoporphyrin a5 monomethyl ester (MgDVP), fucoxanthin, diadinoxanthin and β,β- carotene. Pigment types were based on the following additional pigments: Type 1: chl c1; Type 2: chl c1 and chl c2-Pavlova gyrans-type; Type 3: chl c1 and chl c2-monogalactosyl diacylglyceride ester (chl c2-MGDG [18:4/14:0]); Type 4: chl c1, chl c3 and non-polar chl c1-like; Type 5: chl c1, chl c3, chl c2- MGDG [18:4/14:0] and 4-keto-fucoxanthin; Type 6: chl c3, monovinyl chl c3 (MV-chl c3), chl c2-MGDG [18:4/14:0], 19’-hexanoyloxyfucoxanthin and its 4-keto derivative, and traces of 19’-butanoyloxyfu- coxanthin; Type 7: similar to Type 6, minus MV-chl c3 but with chl c2-MGDG [14:0/14:0] added; Type 8: similar to Type 6, minus MV-chl c3 but with significant 19’-butanoyloxyfucoxanthin. Taxonomic associations ranged from single genera to multiple families – Type 1: Pavlovaceae, Isochrysidaceae and Pleurochrysidaceae; Type 2: Pavlovaceae; Type 3: Isochrysidaceae; Type 4: Prymnesium spp.; Type 5: Ochrosphaera spp.; Type 6: Nöelaerhabdaceae, notably Emiliania spp.; Type 7: Chrysochro- mulina spp.; Type 8: Phaeocystaceae, Prymnesiaceae and Isochrysidaceae. These pigment types showed a strong correlation with available phylogenetic trees, supporting a genetic basis for the pig- ment associations. The additional marker pigments offer oceanographers greater power for detecting haptophytes in mixed populations, while also distinguishing a greater proportion of them from diatoms. KEY WORDS: Haptophyta · HPLC · Chlorophylls c · Fucoxanthins · Pigment types · Phylogeny · Oceanography Resale or republication not permitted without written consent of the publisher INTRODUCTION 2002). They also produce volatile dimethyl sulphide, which produces cloud-condensation nuclei, increasing Haptophyte microalgae are an important component cloud cover and affecting regional climates (Malin et al. of the world’s oceanic phytoplankton (Okada & McIn- 1994). In addition, some species (e.g. Chrysochromulina tyre 1977), blooming seasonally at polar, equatorial and polylepis) are highly toxic to fin-fishes (Moestrup 1994). subtropical latitudes (Brown & Yoder 1994). The calcite- Monitoring of these and other phytoplankton groups is covered coccolithophorids such as Gephyrocapsa essential in order to follow seasonal successions, oceanica and Emiliania huxleyi dominate subtropical impacts of global warming on the marine environment, and sub-polar latitudes (Westbroek et al. 1994), and are and harmful ecological events. significant globally in providing a long-term sink for While taxonomic monitoring of the 200 known hapto- inorganic carbon (Van der Wal et al. 1995, Paasche phyte species by microscopy is possible (Jordan et al. *Corresponding author: Email: [email protected] © Inter-Research 2004 · www.int-res.com 84 Mar Ecol Prog Ser 270: 83–102, 2004 1995, Heimdal 1997), it is so time-consuming that Algal cultures were selected from 7 haptophyte fami- oceanographers routinely use photosynthetic pigment lies — Pavlovaceae, Phaeocystaceae, Prymnesiaceae, profiles as chemotaxonomic markers of phytoplankton Isochrysidaceae, Noëlaerhabdaceae, Pleurochrysida- groups (Jeffrey et al. 1997b). In order to interpret pig- ceae and Hymenomonadaceae — and included many ment data from field samples, however, a thorough globally important species. Multiple isolates of single knowledge of the pigment composition of each of the species or genera from different geographic regions likely species groups of the phytoplankton populations (e.g. Emiliania huxleyi, Phaeocystis antarctica and is necessary. Unfortunately very few wide-ranging pig- Chrysochromulina spp.) were also analysed to deter- ment surveys of algal classes have been published, mine pigment variability. Of the 50 pigments separated, exceptions being for diatoms (Stauber & Jeffrey 1988) 9 chlorophyll c pigments and 5 fucoxanthin derivatives and haptophytes (Jeffrey & Wright 1994). Dominant were useful indicators of 8 haptophyte pigment types. species in field samples should always be assessed This new information shows the diversity of chlorophyll microscopically in representative samples (Andersen et c and fucoxanthin pigments in the photosynthetic appa- al. 1996, Wright & van den Enden 2000). ratus of haptophyte microalgae, and should provide Knowledge of pigment characteristics of any group is useful additional biomarkers for haptophytes in field always limited by the resolution of current separation studies and new clues to photosynthetic mechanisms methods. The haptophyte pigment study of Jeffrey & and phylogenetic relationships. Wright (1994), which used the SCOR-UNESCO HPLC method of Wright et al. (1991), distinguished most of the marker carotenoids, but failed to resolve monovinyl and MATERIALS AND METHODS divinyl analogues of chlorophyll c (e.g. chlorophylls c1 and c2) and additional fucoxanthin derivatives such as 4- Algal cultures. Haptophyte cultures (37 species, 65 keto-19’-hexanoyloxyfucoxanthin (Egeland et al. 2000). strains) were obtained from 3 sources: the CSIRO Algal Nevertheless 4 useful pigment subgroups of the class Culture Collection (Jeffrey & LeRoi 1997, CSIRO 1998), were determined. New advances in HPLC pigment the Australian Antarctic Division, and the Provasoli- technology in the past decade (Jeffrey et al. 1999 Guillard National Centre for Culture of Marine Phyto- [review], Zapata et al. 2000) have allowed a new exam- plankton (CCMP). Strains, isolate information and cul- ination of the pigment composition of this important ture conditions (media and growth temperatures) are group of microalgae in the present work. listed in Table 1. Light irradiances were: 60 to 70 µmol The recent methods of Garrido & Zapata (1997) and quanta m–2 s–1 on 12 h:12 h light:dark cycles (CSIRO 42 –2 –1 Zapata et al. (2000), in which polymeric C18 or mono- strains, CCMP 14 strains) and 40 µmol quanta m s on meric C8 columns were used with pyridine as solvent 16:8 h light:dark cycles (Australian Antarctic Division, modifier, have allowed separation of 11 chlorophyll c 10 strains of Phaeocystis antarctica). pigments (including chlorophylls c1 and c2) across algal Sample preparation. Cultures were examined by classes (Zapata et al. in press) and several new fucoxan- light microscopy before HPLC pigment analysis to thin derivatives. Structural determinations of 2 ‘non- ensure the cells were in excellent health and morphol- polar’ chlorophyll c pigments in Emiliania huxleyi and ogy. Cells were harvested 4 to 6 h into the light cycle Chrysochromulina polylepis showed them to be, not from cultures in exponential growth phase. We filtered phytylated chlorophyll c derivatives (Nelson & Wake- 10 ml of each culture onto 25 mm Whatman GF/F filters ham 1989), but chlorophyll c2-monogalactosyl diacyl- using less than 20 kPa vacuum. Filters were frozen glycerol esters (Garrido et al. 2000, Zapata et al. 2001). immediately at –25°C, and analysed within 12 h. The finding of a chlorophyll attached to a massive lipid Pigment extraction. Frozen filters were extracted side-chain is unique in the photosynthetic literature, under low light in Teflon-lined screw capped tubes and this advance may provide new clues to the photo- with 5 ml 95% methanol using a stainless steel spatula synthetic mechanisms of these important marine species for filter-grinding. The tubes were chilled in a beaker (Jeffrey & Anderson 2000). of ice and sonicated for 5 min in an Ultrasonics Van Lenning et al. (2003) recently used the Zapata et Australia bath. Extracts were then filtered through al. (2000) technique to study the pigment content of 9 25 mm diameter hydrophilic Teflon (PTFE) syringe species of Pavlovaceae, finding 3 pigment types that filters (MFS HP020, 0.2 µm pore size) to remove cell corresponded with phylogenetic relationships (based on and filter debris. An aliquot (0.5 ml) of the methanol 18S rDNA) and morphological differences within the extract was mixed with 0.2 ml of water and 200 µl was family. injected immediately into the HPLC. This procedure In this paper, we re-examine the photosynthetic pig- avoids peak distortion of early eluting peaks (Zapata & ments of haptophyte cultures from 7 families (37 spe- Garrido 1991) and prevents the loss of non-polar cies; 65 strains) using the HPLC methods cited above. pigments prior to injection. Zapata et al.: Photosynthetic pigments in Haptophyta 85 Table 1. Haptophyte species, strain codes, culture media and growth temperatures of 37 species (65 strains) examined. CS = CSIRO Culture Collection of Living Microalgae,